干旱胁迫对管花肉苁蓉组织培养体系中苯乙醇苷类成分含量的影响
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摘要
目的建立管花肉苁蓉Cistanche tubulosa组织培养体系,并考察干旱胁迫对其中苯乙醇苷类成分含量的影响。方法通过液质联用技术分析管花肉苁蓉愈伤组织与悬浮培养体系中的主要化学成分,绘制其生长曲线,并通过渗透压调节剂(NaCl、甘露醇和PEG6000)模拟干旱胁迫环境,考察组织培养体系中松果菊苷和毛蕊花糖苷含量的变化。结果管花肉苁蓉愈伤组织与悬浮培养细胞均能生成松果菊苷和毛蕊花糖苷;绘制管花肉苁蓉愈伤组织生长曲线,确定30d为最佳继代时间;NaCl和甘露醇所诱导的干旱胁迫不利于愈伤组织细胞的生长及其苯乙醇苷类成分的积累,而6%PEG6000能显著促进悬浮细胞中苯乙醇苷类化合物的生成,诱导15d后,松果菊苷和毛蕊花糖苷的生物量分别为(1.07±0.10)g/L和(0.12±0.01)g/L,分别可达细胞干质量的20.94%和2.27%,为对照组的1.29倍和1.19倍。结论 PEG6000介导的干旱胁迫对管花肉苁蓉悬浮细胞中松果菊苷和毛蕊花糖苷的积累有明显促进作用。
Objective To establish the plant tissue culture system of Cistanche tubulosa, and determine the effect of drought stress on accumulation of two respective phenylethanoid glycosides in it. Methods The major chemical constituents of C. tubulosa by tissue culture were analyzed by HPLC-UV and HR-MS. The cell growth curves were also determined. In addition, the effects of drought stress on the phenylethanoid glycosides(echinacoside and acteoside) content in the tissue culture system of C. tubulosa were also studied by using Na Cl, mannitol and PEG6000 as osmotic regulators, respectively. Results Chemical constituents analyses revealed that callus and suspension cultures of C. tubulosa could produce the respective phenylethanoid glycosides of echinacoside and acteoside as in wild plant;Cell growth curves indicated that 30 d were the optimum culture period of callus culture; The cell growth rate and the accumulation of echinacoside and acteoside were mostly inhibited when the callus cells were under drought stress induced by Na Cl or mannitol.Meanwhile, the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa could be effectively enhanced by treatment with PEG6000. The maximum biomass of echinacoside and acteoside could reach to(1.07 ± 0.10) g/L and(0.12 ± 0.01) g/L 15 d after induction, respectively. And their contents were 20.94% and 2.27% separately based on the cell dry weight(DW) after 15 d of treatment with 6% PEG6000, which were 1.29 and 1.19 fold higher than the control group. Conclusion Drought stress induced by PEG6000 could effectively enhance the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa.
Objective To establish the plant tissue culture system of Cistanche tubulosa, and determine the effect of drought stress on accumulation of two respective phenylethanoid glycosides in it. Methods The major chemical constituents of C. tubulosa by tissue culture were analyzed by HPLC-UV and HR-MS. The cell growth curves were also determined. In addition, the effects of drought stress on the phenylethanoid glycosides(echinacoside and acteoside) content in the tissue culture system of C. tubulosa were also studied by using Na Cl, mannitol and PEG6000 as osmotic regulators, respectively. Results Chemical constituents analyses revealed that callus and suspension cultures of C. tubulosa could produce the respective phenylethanoid glycosides of echinacoside and acteoside as in wild plant;Cell growth curves indicated that 30 d were the optimum culture period of callus culture; The cell growth rate and the accumulation of echinacoside and acteoside were mostly inhibited when the callus cells were under drought stress induced by Na Cl or mannitol.Meanwhile, the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa could be effectively enhanced by treatment with PEG6000. The maximum biomass of echinacoside and acteoside could reach to(1.07 ± 0.10) g/L and(0.12 ± 0.01) g/L 15 d after induction, respectively. And their contents were 20.94% and 2.27% separately based on the cell dry weight(DW) after 15 d of treatment with 6% PEG6000, which were 1.29 and 1.19 fold higher than the control group. Conclusion Drought stress induced by PEG6000 could effectively enhance the accumulation of echinacoside and acteoside in cell suspension culture of C. tubulosa.
引文
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[15]Ouyang J,Wang X D,Zhao B,et al.Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media[J].Biotechnol Lett,2003,25(3):223-225.
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[17]Liu C Z,Cheng X Y.Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of Cistanche deserticola by osmotic stress[J].Plant Cell Rep,2008,27(2):357-362.
[18]Hu G S,Jia J M,Kim D H.Effects of feeding tyrosine and phenylalanine on the accumulation of phenylethanoid glycosides to Cistanche deserticola cell suspension culture[J].Chin J Nat Med,2014,12(2):367-372.
[19]Ouyang J,Wang X D,Zhao B,et al.Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola[J].Process Biochem,2005,40(11):3480-3484.
[20]Lu C T,Mei X G.Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola[J].Biotechnol Lett,2003,25(17):1437-1439.
[21]Song Y L,Song Q Q,Li J,et al.An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C.tubulosa using high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry[J].J Chromatogr A,2016,1429:238-247.
[22]Liu X,Yan Y R,Liu Y Y,et al.Cell culture establishment and regulation of two phenylethanoid glycosides accumulation in cell suspension culture of desert plant Cistanche tubulosa[J].Plant Cell Tissue Organ,2018,134(1):107-118.
[23]Ehsanpour A A,Amini F.Effect of salt and drought stress on acid phosphatase activities in alfalfa(Medicago sativa L.)explants under in vitro culture[J].Afr J Biotechnol,2003,2(5):133-135.
[24]Tonon G,Kevers C,Faivre-Rampant O,et al.Effect of NaCl and mannitol iso-osmotic stresses on proline and free polyamine levels in embryogenic Fraxinus angustifolia callus[J].J Plant Physiol,2004,161(6):701-708.
[25]王兰兰,张立军,陈贵,等.甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比[J].生态学杂志,2006,12(12):1508-1514.
[26]曾丽兰,林玉玲,王亚婷,等.渗透胁迫对龙眼胚性愈伤组织SOD活性的影响[J].福建农林大学学报:自然科学版,2014,43(1):14-19.
[27]Kim S I,Choi H K,Kim J H.Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis[J].Enzyme Microb Tech,2001,28(2/3):202-209.
[28]盛东峰,陈龙.聚乙二醇-6000胁迫对丹参毛状根中丹参酮积累的影响[J].中草药,2013,44(9):1181-1185.
[2]Jiang Y,Tu P F.Analysis of chemical constituents in Cistanche species[J].J Chromatogr A,2009,1216(11):1970-1979.
[3]雷丽,宋志宏,屠鹏飞.肉苁蓉属植物的化学成分研究进展[J].中草药,2003,34(5):473-476.
[4]宋志宏,屠鹏飞,赵玉英,等.管花肉苁蓉的苯乙醇苷类成分[J].中草药,2000,31(11):808-810.
[5]Tu P F,Song Z H,Jiang Y,et al.Chemical constituents of Cistanche sinensis[J].J Asian Nat Prod Res,2007,9(1):79-84.
[6]Tu P F,Song Z H,Shi H M,et al.Aryethyl glycosides and oligosaccharide from the stem of Cistanche tubulosa[J].Helv Chim Acta,2006,89(5):927-935.
[7]Tu P F,Shi H M,Song Z H,et al.Chemical constituents from Cistanche sinensis(Orobanchaceae)[J].Biochem Syst Ecol,2013,47:21-24.
[8]Zhao Q,Gao J P,Li W W,et al.Neurotrophic and neurorescue effects of echinacoside in the subacute MPTP mouse model of Parkinson’s disease[J].Brain Res,2010,1346:224-236.
[9]Xue D J,Zhang M,Wu X H,et al.Studies on the active antisenile constituents in Cistanche deserticola Y.C.Ma.[J].Plant Med,1995,20(11):687-704.
[10]Piao H,Kamiya N,Hirata A,et al.A novel solid-in-oil nanosuspension for transdermal delivery of diclofenac sodium[J].Pharm Res,2008,25(4):896-901.
[11]Zhao J,Liu T,Ma L,et al.Protective effect of acteoside on immunological liver injury induced by Bacillus Calmette-Guerin plus lipopolysaccharide[J].Plant Med,2009,75(14):1463-1469.
[12]Nour-Eddine E S,Albert K,Samira K,et al.Anti-oxidantive effect of compounds isolated from Globularia alypum L.structure-activity relationship[J].Lwt-food Sci Technol,2007,40(7):1246-1252.
[13]皋聪,王传社,屠鹏飞,等.苁蓉总苷对血管性痴呆大鼠学习记忆的影响及机制研究[J].中草药,2005,36(12):1852-1855.
[14]Chen C H,Song T Y,LiangY C,et al.Acteoside and6-O-acetylacteoside downregulate cell adhesion molecules induced by IL-1βthrough inhibition of ERKand JNK in human vascular endothelial cells[J].J Agric Food Chem,2009,57(19):8852-8859.
[15]Ouyang J,Wang X D,Zhao B,et al.Formation of phenylethanoid glycosides by Cistanche deserticola callus grown on solid media[J].Biotechnol Lett,2003,25(3):223-225.
[16]Hu G S,Hur Y J,Jia J M,et al.Effects of2-aminoindan-2-phosphonic acid treatment on the accumulation of salidroside and four phenylethanoid glycosides in suspension cell culture of Cistanche deserticola[J].Plant Cell Rep,2001,30(4):665-674.
[17]Liu C Z,Cheng X Y.Enhancement of phenylethanoid glycosides biosynthesis in cell cultures of Cistanche deserticola by osmotic stress[J].Plant Cell Rep,2008,27(2):357-362.
[18]Hu G S,Jia J M,Kim D H.Effects of feeding tyrosine and phenylalanine on the accumulation of phenylethanoid glycosides to Cistanche deserticola cell suspension culture[J].Chin J Nat Med,2014,12(2):367-372.
[19]Ouyang J,Wang X D,Zhao B,et al.Enhanced production of phenylethanoid glycosides by precursor feeding to cell culture of Cistanche deserticola[J].Process Biochem,2005,40(11):3480-3484.
[20]Lu C T,Mei X G.Improvement of phenylethanoid glycosides production by a fungal elicitor in cell suspension culture of Cistanche deserticola[J].Biotechnol Lett,2003,25(17):1437-1439.
[21]Song Y L,Song Q Q,Li J,et al.An integrated strategy to quantitatively differentiate chemome between Cistanche deserticola and C.tubulosa using high performance liquid chromatography-hybrid triple quadrupole-linear ion trap mass spectrometry[J].J Chromatogr A,2016,1429:238-247.
[22]Liu X,Yan Y R,Liu Y Y,et al.Cell culture establishment and regulation of two phenylethanoid glycosides accumulation in cell suspension culture of desert plant Cistanche tubulosa[J].Plant Cell Tissue Organ,2018,134(1):107-118.
[23]Ehsanpour A A,Amini F.Effect of salt and drought stress on acid phosphatase activities in alfalfa(Medicago sativa L.)explants under in vitro culture[J].Afr J Biotechnol,2003,2(5):133-135.
[24]Tonon G,Kevers C,Faivre-Rampant O,et al.Effect of NaCl and mannitol iso-osmotic stresses on proline and free polyamine levels in embryogenic Fraxinus angustifolia callus[J].J Plant Physiol,2004,161(6):701-708.
[25]王兰兰,张立军,陈贵,等.甘薯愈伤组织对干旱胁迫和盐胁迫的生理反应对比[J].生态学杂志,2006,12(12):1508-1514.
[26]曾丽兰,林玉玲,王亚婷,等.渗透胁迫对龙眼胚性愈伤组织SOD活性的影响[J].福建农林大学学报:自然科学版,2014,43(1):14-19.
[27]Kim S I,Choi H K,Kim J H.Effect of osmotic pressure on paclitaxel production in suspension cell cultures of Taxus chinensis[J].Enzyme Microb Tech,2001,28(2/3):202-209.
[28]盛东峰,陈龙.聚乙二醇-6000胁迫对丹参毛状根中丹参酮积累的影响[J].中草药,2013,44(9):1181-1185.